Potentiation of the Microbicide 2-(Thiocyanomethylthio)benzothiazole using an n-alkyl heterocyclic compound

ABSTRACT

Microbicidal compositions are described. The compositions comprise (a) 2-(Thiocyanomethylthio)benzothiazole (TCMTB) and (b) an N-alkyl heterocyclic compound of the formula: 
                 
 
The variable “n” ranges from 5 to 17, and the heterocyclic ring defined by 
                 
 
is a substituted or unsubstituted ring having four to eight members. Components (a) and (b) are present in a combined amount effective to control the growth of at least one microorganism. Methods for controlling the growth of microorganisms on various substrates or in aqueous systems are also described. Also described is the industrial application of the microbicidal composition in the leather industry, the lumber industry, the papermaking industry, the textile industry, the agricultural industry, and the coating industry, as well as in aqueous systems.

CONTINUING DATA

This application is a continuation of application Ser. No. 08/456,098,filed May 30, 1995, now U.S. Pat. No. 5,693,631.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions for controlling the growthof microorganisms on a variety of substrates and in aqueous systems.More particularly, the invention relates to combinations of2-(Thiocyanomethylthio) benzothiazole (TCMTB) with an N-alkylheterocyclic compound and the use of such combinations as microbicides.

BACKGROUND OF THE INVENTION

A large variety of commercial, industrial, agricultural, and woodmaterials or products are subject to microbiologicl attack ordegradation which reduces or destroys their economic value. Examples ofsuch materials or products include surface coatings, lumber, seeds,plants, leather and plastics. The various temperatures at which suchmaterials or products are manufactured, stored, or used as well as theirintrinsic characteristics make them susceptible to growth, attack, anddegradation by common microorgansims such as algae, fungi, yeasts, andbacteria. These microorganisms may be introduced during a manufacturingor other industrial process, by exposure to air, tanks, pipes,equipment, and humans. They can also be introduced while using amaterial or product, for example, by multiple openings and reclosures ofpackages or from stirring or removing material with contaminatedobjects.

Aqueous systems are also highly subject to microbiological growth,attack, and degradation. The aqueous system may be a fresh, brackish orsaltwater system. Exemplary aqueous systems include, but are not limitedto, latexes, surfactants, dispersants, stabilizers, thickeners,adhesives, starches, waxes, proteins, emulsifying agents, celluloseproducts, metal working fluids, cooling water, waste water, aqueousemulsions, aqueous detergents, coating compositions, paint compositions,and resins formulated in aqueous solutions, emulsions or suspensions.These systems frequently contain relatively large amounts of water andorganic material causing them to be environments well-suited formicrobiologic growth and thus attack and degradation.

Microbiological degradation of aqueous systems may manifest itself as avariety of problems, such as loss of viscosity, gas formation,objectionable odors, decreased pH, emulsion breaking, color change, andgelling. Additionally, microbiological deterioration of aqueous systemscan cause fouling of the related water-handling system, which mayinclude cooling towers, pumps, heat exchangers, and pipelines, heatingsystems, scrubbing systems, and other similar systems.

Another objectionable phenomenon occurring in aqueous systems,particularly in aqueous industrial process fluids, is slime formation.Slime formation can occur in fresh, brackish or salt water systems.Slime consists of matted deposits of microorganisms, fibers and debris.It may be stringy, pasty, rubbery, tapioca-like, or hard, and may have acharacteristic undesirable odor that is different from that of theaqueous system in which it formed. The microorganisms involved in itsformation are primarily different species of spore-forming andnonspore-forming bacteria, particularly capsulated forms of bacteriawhich secrete gelatinous substances that envelop or encase the cells.Slime microorganisms also include filamentous bacteria, filamentousfungi of the mold type, yeast, and yeast-like organisms. Slime reducesyields in production and causes plugging, bulking, and other problems inindustrial water systems.

Various chemicals known as industrial microbicides have been used toprevent microbiological deterioration of industrial systems, rawmaterials, and products. For instance,2-(Thiocyanomethylthio)benzothiazole (TCMTB) is a well known, effectivemicrobicide available for such uses. The use of TCMTB as an industrialmicrobicide has been described in U.S. Pat. Nos. 4,293,559, 4,866,081,4,595,691, 4,944,892, 4,893,373, and 4,479,961. TCMTB is manufactured byBuckman Laboratories International Inc., and sold as Busan® 30L product,Busan® 3WB product, Busan® 1030 product, Busan® 1118 product as well asother products. TCMTB has the following chemical structure:

Despite the existence of such microbicides, industry is constantlyseeking more cost-effective technology which offers equal or betterprotection at lower cost and lower concentration. The concentration ofconventional microbicides and the corresponding treatment costs for suchuse, can be relatively high. Important factors in the search forcost-effective microbicides include the duration of microbicidal effect,the ease of use, and the effectiveness of the microbicide per unitweight.

SUMMARY OF THE INVENTION

In view of industry's search for more cost effective microbicides, thepresent invention offers an improvement over current products orpractices.

A first embodiment of the invention provides a microbicidal composition.This compostion comprises (a) 2-(Thiocyanomethylthio )benzothiazole(TCMTB) as a microbicide, and (b) an N-alkyl heterocyclic compound ofthe formula:

For the N-alkyl heterocyclic compound, n may vary from 5 to 17 and theheterocyclic ring defined by

may be a substituted or unsubstituted ring having four to eight members.In the microbicidal composition, the TCMTB (a) and the N-alkylheterocyclic compound (b) are present in a combined amount effective tocontrol the growth of at least one microorganism. The combination ofTCMTB with an N-alkyl heterocyclic compound achieves superiormicrobicidal activity at lower concentrations and lower cost than TCMTBalone against microbiological attack or degradation such as discussedabove.

Another embodiment of the present invention provides a method forcontrolling the growth of a microorganism on a substrate. This methodcomprises contacting the substrate with TCMTB and an N-alkylheterocyclic compound, having the above formula. The TCMTB and N-alkylheterocyclic compound are present in a combined amount effective tocontrol the growth of at least one microorganism on the substrate.

Another embodiment of the invention provides a method for controllingthe growth of microorganisms in an aqueous system capable of supportinggrowth of a microorganism. This method comprises the step of treatingthe aqueous system with 2-(Thiocyanomethylthio )benzothiazole (TCMTB)and an N-alkyl heterocyclic compound of the above formula, where theTCMTB and the N-alkyl heterocyclic compound are present in a combinedamount effective to control the growth of at least one microorganism inthe aqueous system.

The combination of TCMTB and an N-alkyl heterocyclic compound accordingto the invention is useful in preventing the microbiological attack,degradation, or deterioration of various types of raw materials andproducts such as leather, textiles, pulp, paper and paperboard,coatings, lumber, as well as agricultural products such as seeds andcrops. Advantageously, the combination may be used in various industrialprocesses used to prepare or manufacture these products. Accordingly,additional embodiments of the present invention employ the combinationto control the growth of microorganisms on or in such industrialproducts, raw materials or processes.

The foregoing and other features and advantages of the present inventionwill be made more apparent from the following detailed description andpreferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to a microbicidalcomposition comprising 2-(Thiocyanomethylthio)benzothiazole (TCMTB) as amicrobicide and an N-alkyl heterocyclic compound. Mixtures of N-alkylheterocyclic compounds may be used. The TCMTB and the N-alkylheterocyclic compound are present in a combined amount effective tocontrol the growth of at least one microorgamsm.

The N-alkyl heterocyclic compounds employed in the present inventionhave the following general formula:

The variable “n” may vary from 5 to 17, and preferably from 9 to 15.Most preferably, n is 11. The alkyl chain defined by CH₃C_(n)H_(2n)— maybe branched or unbranched. Branched alkyl chains may lose some of theirsolubility in water or other aqueous systems. Unbranched alkyl groupsare generally preferred.

The heterocyclic ring defined by

may have four to eight members and is preferably a five-, six-, seven-,or eight-member ring. Most preferably the heterocyclic ring is asix-membered ring.

Although the heterocyclic ring always contains one nitrogen atom, theremainder is generally a carbocycle. However, the ring may contain oneor more additional heteroatoms selected from N, O, or S. The ring may besaturated or unsaturated. The ring may also have common substituentssuch as alkyl groups, substituted alkyl groups, alkenyl groups,substituted alkenyl groups, amino groups, an oxo group to form a cyclicketone, halogens, etc. The heterocyclic ring may also be part of amultiple ring structure.

The heterocycles listed below exemplify substituted or unsubstitutedheterocyclic rings which may be used in the N-alkyl heterocycliccompounds utilized in preferred embodiments of the present invention.Examples of five-membered heterocyclic rings include, but are notlimited to, pyrrolidinyl, 2-pyrrolidinonyl, pyrrolinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, imidazolidinyl, imidazolinyl, imidazolyl andoxazolidinonyl. Six-membered rings include, but are not limited to,piperidinyl, piperazinyl, and morpholinyl. Seven- and eight-memberedrings such as hexamethyleneiminyl and heptamethyleneiminyl may also beused in the present invention. One of ordinary skill will appreciatethat other heterocyclic rings may also be used.

N-alkyl heterocyclic compounds useful in the invention are availableeither commercially from chemical supply houses or may be prepared fromstarting materials using well-known literature methods. U.S. Pat. No.5,250,194 discloses exemplary methods and is incorporated herein byreference.

U.S. Pat. No. 5,250,194 also describes N-dodecyl heterocyclic compoundsand their use as microbicides for aqueous systems to inhibit the growthof microorganisms, the formation of slime in aqueous systems, or thedisfigurement or deterioration of substances susceptible tomicrobiological growth. One example of an N-alkyl heterocyclic compounduseful as such a microbicide is N-dodecyl morpholine (DDM). DDM ismanufactured by BASF GmbH and by Buckman Laboratories InternationalInc., Memphis, Tenn.

Preferred N -alkyl heterocyclic compounds for use in the presentinvention include N-dodecyl morpholine, N-dodecyl imidazole, N-dodecyl-2,6-dimethyl-morpholine,N-dodecyl-5-chloromethyl-2-oxazolidinone, N-dodecyl-2-pyrrolidinone,N-dodecyl hexamethyleneimine, N-dodecyl pyrrolidine,N-dodecyl-3-methyl-piperidine, N-dodecyl piperidine,N-dodecyl-4-methyl-piperidine and N-dodecyl-2-methyl-piperidine. Mostpreferred of these compounds are N-dodecyl morpholine, (DDM), andN-dodecyl imidazole, (DDI).

Depending on the application, microbicidal compositions according to thepresent invention may be prepared in various forms known in the art. Forexample, the composition may be prepared in liquid form as an aqueoussolution, dispersion, emulsion, or suspension, a dispersion orsuspension in a non-solvent, or as a solution by dissolving the TCMTBand the N-alkyl heterocyclic compound in a solvent or combination ofsolvents. Suitable solvents include, but are not limited to, methylethers of glycols, M-pyrol or petroleum distillates. The microbicidalcomposition may be prepared as a concentrate for dilution prior to itsintended use. Common additives such as surfactants, emulsifiers,dispersants, and the like may be used as known in the art to increasethe solubility of the TCMTB or N-alkyl heterocyclic compound in a liquidcomposition or system, such as an aqueous composition or system. In manycases, the biocidal composition of the invention may be solubilized bysimple agitation.

Microbicidal compositions of the present invention may also be preparedin solid form, for example as a powder or tablet, using means known inthe art. In a preferred method of preparation, a liquid productcontaining TCMTB is deposited on a carrier such as diatomaceous earth orkaolin and mixed with an N-alkyl heterocyclic compound in the form of aliquid or solution to form a powder or tablet.

The TCMTB and the N-alkyl heterocyclic compound may be combined in asingle composition. Alternatively, the TCMTB and the N-alkylheterocyclic compound may be employed as separate components such thatcombined amount for the intended use is effective to control the growthof at least one microorganism.

As mentioned above, a microbicidal composition of the inventiondemonstrates an unexpected enhanced microbicidal effect between therespective components, TCMTB and an N-alkyl heterocyclic compound. Thatis, the combination of TCMTB and an N-alkyl heterocyclic compoundachieves superior microbicidal activity at lower concentrations tocontrol the growth of microorganisms as compared to the microbicidalcapability of TCMTB alone. Thus, the N-alkyl heterocyclic compoundpotentiates, or even synergistically enhances, the microbicidal effectof the TCMTB. Such a superior effect presents a distinct economicadvantage and increases the microbicide's effectiveness per unit weight.

According to the present invention, control of the growth of amicroorganism on a substrate or in an aqueous system means control to,at, or below a desired level and for a desired period of time for theparticular substrate or system. This can vary from the completeprevention or inhibition of microbiological growth to control at acertain desired level and for a desired time. The combination of TCMTBand N-alkyl heterocyclic compound described here can, in many cases,reduce the total microbiological count to undetectable limits andmaintain the count at that level for a significant period of time.Accordingly, the combination may be used to preserve a substrate orsystem.

The effective amount or percentage of the combination of TCMTB and anN-alkyl heterocyclic compound necessary to achieve the desired resultwill vary somewhat depending on the substrate or aqueous system to beprotected, the conditions for microbial growth, and the degree ofprotection desired. For a particular application, the amount of choicemay be determined by routine testing of various amounts prior totreatment of the entire affected substrate or system. In general, aneffective amount used on a substrate ranges from about 0.0001% to about4% (w/w); preferably about 0.0001% to about 0.2%. With aqueous systems,an effective amount may range from about 0.5 to about 5000 parts permillion, more preferably from about 5 to about 1000 parts per million ofthe aqueous system, and most preferably from, about 10 to about 25 partsper million. Similar amounts effectively control slime formation. Forslime control, effective amounts preferably range from about 1 to about200 parts per million, and more preferably, from about 1 to about 25parts per million of the aqueous system.

In a preferred embodiment, combinations of TCMTB and an N-alkylheterocyclic compound are those combinations having a weight ratio ofTCMTB to N-alkyl heterocyclic compound from about 99:1 to about 1:99.More preferably the weight ratio is from about 60:10 to about 10:60, andmost preferably, from about 50:50 to about 25:75. The weight ratio mayvary depending on the intended use, the microorganism encountered aswell as the particular material, product, or system to which thecombination according to the invention is applied.

The combination of TCMTB and an N-alkyl heterocyclic compound may beapplied in a variety of industrial uses and processes for microorganismcontrol. The combination may be used in place of and in the same manneras other microbicides traditionally used in the particular industry. Asdiscussed above, such industries include, but are not limited to theleather industry, the lumber industry, the papermaking industry, thetextile industry, the agricultural industry, and the coating industry.The combination of TCMTB and an N-alkyl heterocyclic compound may alsobe used with aqueous systems such as those previously discussed whichare subject to microbiological attack and degradation. The problemscaused by microbiological attack and deterioration in these variousapplications has been described above. The use of the combination ofTCMTB and an N-alkyl heterocyclic compound according to the invention tocontrol the growth of microorganisms in particular exemplaryapplications is described below.

The invention also relates to a method for controlling the growth ofmicroorganisms on various substrates. The method comprises the step ofcontacting a substrate susceptible to microbiological growth or attackwith TCMTB and an N-alkyl heterocyclic compound, as described above. TheTCMTB and N-alkyl heterocyclic compound are present in a combined amounteffective to control the growth of at least one microorganism on thesubstrate. Preferably, the method may be used to eliminate or preventsubstantially all microbiological growth on the substrate. As discussedabove, the TCMTB and the N-alkyl heterocyclic compound may be appliedtogether or as separate compositions. Preferred applications of thisgeneral method are discussed below.

In the leather industry, the combination of TCMTB and an N-alkylheterocyclic compound may be used to control the growth ofmicroorganisms on a hide during a tanning process. To achieve thiscontrol, the hide is contacted with a combined amount of TCMTB and anN-alkyl heterocyclic compound effective to control the growth of atleast one microorganism on the hide. The combination of the TCMTB andthe N-alkyl heterocyclic compound may be used in the tanning process insimilar amounts and manner similar to that used to apply othermicrobicides used in the tanning industry. The type of hide may be anytype of hide or skin that is tanned, for example cowhide, snake skin,alligator skin, sheep skin, and the like. The amount used, to someextent, will depend on the degree of microbiological resistance requiredand may be readily determined by one skilled in the art.

A typical tanning process comprises a number of stages, including, butnot limited to, a pickling stage, a chrome-tanning stage, avegetable-tanning stage, a post-tan washing stage, a retanning stage, adyeing stage, and a fatliquoring stage. The combination of TCMTB and anN-alkyl heterocyclic compound may be used during all process stages inthe tanning process in addition to those stages where a knownmicrobiological problem is occurring. In each stage, the combination ofTCMTB and an N-alkyl heterocyclic compound may be a component of theappropriate tanning liquor applied to the hide undergoing tanning.

Incorporating the TCMTB and an N-alkyl heterocyclic compound in atanning liquor protects the hide from microbiological deteriorationduring the tanning process. Preferably, the combination is uniformlydispersed, e.g., under agitation, into an appropriate liquor to be usedin a tanning process. Typical tanning liquors include, for example, apickling liquor, a chrome-tanning liquor, a vegetable-tanning liquor, apost-tan washing liquor, a retanning liquor, a dye liquor, and afatliquor. This method of application ensures that the combinationapplied to the hides protects against microbiological attack,deterioration, or other microbiological degradation.

In a somewhat analogous nature, the combination of the invention mayalso be employed to control the growth of microorganisms on a textilesubstrate in a textile manufacturing process. Contacting the textilesubstrate with a combination of TCMTB and an N-alkyl heterocycliccompound according to the invention effectively controls the growth of amicroorganism on the textile substrate. In a textile process, thecombination may be used in similar amounts and a manner similar to othermicrobicides commonly used in such processes. As one of ordinary skillwould appreciate, particular amounts generally depend on the textilesubstrate and the degree of microbiological resistance required.

The step of contacting the textile substrate with the combination ofTCMTB and an N-alkyl heterocyclic compound may be accomplished usingmeans known in the textile art. To control microbiological growth, atextile process generally dips the textile substrate into a bathcontaining a microbicide, alone or with other chemicals used to treatthe textile substrate. Alternatively, the textile substrate may besprayed with a formulation containing a microbicide. In the bath or thespray, the combination of TCMTB and N-alkyl heterocyclic compoundaccording to the invention are present in a combined amount effective tocontrol the growth of at least one microorganism on the textilesubstrate. Preferably, the bath and the spray are aqueous-basedcompositions.

To preserve the value of its raw materials and products, the lumberindustry also must control the growth of microorganisms in order toprevent microbiological degradation. The combination of TCMTB and anN-alkyl heterocyclic compound according to the invention is effective tocontrol the growth of microorganisms on lumber.

The combination of TCMTB and an N-alkyl heterocyclic compound may beused to protect the lumber in similar amounts and a similar manneremployed for other microbicides used in the lumber industry. Contactinglumber with an effective amount of the combination may be accomplished,for example, by spraying the lumber with an aqueous formulationcontaining the combination of TCMTB and an N-alkyl heterocycliccompound, by dipping the lumber into a dip bath containing thecombination, or other means known in the art. Dipping the lumber in anaqueous bath is preferred.

The TCMTB and the N-alkyl heterocyclic compound are preferably uniformlydispersed in a bath (for example, by agitation) prior to the dipping ofthe lumber into the bath. In general, the lumber is dipped into thebath, raised, allowed to drip dry, and then air dried. The dip time willdepend, as is known in the art, on a variety of factors such as thedegree of microbiological resistance desired, the moisture content ofthe lumber, type and density of the wood, etc. Pressure may be appliedto promote penetration of the combination into the lumber being treated.Applying a vacuum to the upper surface of the lumber may also be used todegas the lumber and promote increased wetting of the lumber by a bathcontaining the microbicidal combination.

The combination of TCMTB and an N-alkyl heterocyclic compound accordingto the invention also has uses in the agricultural industry. To controlthe growth of microorganisms on a seed or plant, the seed or plant maybe contacted with TCMTB and an N-alkyl heterocyclic compound in acombined amount effective to control the growth of at least onemicroorganism on the seed or plant. This contacting step may beaccomplished using means and amounts known in the agricultural industryfor other microbicides. For example, the seed or plant may be sprayedwith an aqueous formulation containing the combination of TCMTB andN-alkyl heterocyclic compound, or dipped into a bath containing thecombination. After being sprayed or dipped, the seed or plant isgenerally dried by means known in the art such as drip drying, heateddrying, or air drying. For plants or crops, the combination may also beapplied using a soil drench. Soil drenching is particularly advantageouswhen the microorganisms of concern inhabit the soil surrounding theplant.

Yet another aspect of the present invention is a method for controllingthe growth of microorganisms in an aqueous system capable of supportingsuch growth. The aqueous system is treated with TCMTB and an N-alkylheterocyclic compound such that the TCMTB and N-alkyl heterocycliccompound are present in a combined amount effective to control thegrowth of at least one microorganism in the aqueous system. Thisincludes controlling, and preferably preventing, slime formation in theaqueous system.

Examples of various aqueous systems include, but are not limited to,latexes, surfactants, dispersants, stabilizers, thickeners, adhesives,starches, waxes, proteins, emulsifying agents, cellulose products,aqueous emulsions, aqueous detergents, coating compositions, paintcompositions, alum compositions, and resins formulated in aqueoussolutions, emulsions or suspensions. The combination may also beemployed in aqueous systems used in industrial processes such as metalworking fluids, cooling waters (both intake cooling water and effluentcooling water), and waste waters including waste waters or sanitationwaters undergoing treatment of the waste in the water, e.g. sewagetreatment.

As with the other uses discussed above, the combination of the inventionmay be used in the same amounts and in the same manner as microbicidestraditionally used in these various aqueous systems. The combination notonly protects the aqueous system prior to use or when stored, but inmany cases protects the aqueous system when in use or in appropriateapplications even after the aqueous system has dried. When used in apaint formulation for example, the combination not only protects thepaint in the can, but also the paint film after being applied to asubstrate.

Another embodiment of the present invention is a method for controllingthe growth of microorganisms on paper or in a papermaking process, e.g.,in a pulp or paper slurry and on a finished paper product such as paperboard. The paper, pulp, or slurry is contacted with TCMTB and an N-alkylheterocyclic compound in a combined amount effective to control thegrowth of at least one microorganism on the paper, the pulp or in aslurry. The contacting step is accomplished using means and amountsknown in the papermaking art.

According to this aspect of the invention, for example, a forming web ona papermaking machine (or a wet-lap pulp) may be contacted with thecombination of TCMTB and an N-alkyl heterocyclic compound by spraying anaqueous dispersion containing the TCMTB and N-alkyl heterocycliccompound onto the pulp after the pulp leaves the presses in apapermaking process. Or, the TCMTB and the N-alkyl heterocyclic compoundmay be incorporated into a bath used at the wet or size press and theweb contacted by nipping the web to incorporate the combination into theweb with any other agents applied at the press. Alternatively, the pulpmay be contacted by mixing the TCMTB and N-alkyl heterocyclic compoundinto the pulp/white water mixture, preferably prior to the pulp reachingthe formation wire.

When treating paper (which includes paperboard and other cellulosicproducts or substrates), the TCMTB and N-alkyl heterocyclic compound maybe added into pulp slurries in the headbox, in the substrate formingsolution, or in the white water system to treat the water system itselfor for incorporation into the body of the paper. Alternatively, as withother known microbicides, the combination of TCMTB and an N-alkylheterocyclic compound according to the invention may be mixed into acoating used to coat the finished paper.

The activity of the combinations described above has been confirmedusing standard laboratory techniques as discussed below. In many cases,the N-alkyl heterocyclic compound potentiates, or even synergisticallyenhances, the microbicidal affect of the TCMTB.

The following examples are intended to illustrate, not limit, thepresent invention.

EXAMPLE 1

Fungicidal Activity of TCMTB and an N-Alkyl Heterocyclic Compound

The fungicidal effects of combinations use of TCMTB and N-alkylheterocyclic compounds against fungi were measured by the binarydilution method. Combinations of TCMTB and N-dodecyl morpho line (DDM)were tested against a mixture of fungal species isolated from moldedchrome-tanned leather. Busan® 30L product, a 30% TCMTB formulation, andBL 2180 product, a 60% formulation of DDM, were used in these tests.Both the Busan® 30L product and BL 2180 product are available fromBuckman Laboratories Inc., Memphis, Tenn.

The original Trichoderma culture used in the testing was isolated andcultured from a molded sample of chrome-tanned or wet blue leather. Froma suspension of Trichoderma viride spores, 0.1 ml of this suspension wasadded to each of 200 standard sized test tubes. Each test tube contained4.5 ml of nutrient salts broth. The test tubes were then agitated toensure proper mixing of the fungal spores into the nutrient broth. Onceinoculated and treated at various concentrations and combinations, thetest tubes were incubated at 28° C. for 7 days. The lowest concentrationof each chemical compound or combination of compounds that prevented thegrowth of the fungi in the broth, i.e., the minimum inhibitoryconcentration, was taken as the end-point.

Synergism was demonstrated using the method devised by Kull, E. C.,Eisman, P. C., Sylwestrwicz, H. D., and Mayer, R. L., AppliedMicrobiology, 1961, pp.538-541. Kull et al. apply the followingsummation (synergism index) to determine whether a synergistic effectwas achieved:QA/Qa+QB/QbIn this formula QA, Qa, QB, and Qb have the following values:

Qa=Concentration of compound A in parts per million, acting alone, whichproduced an end point. This represents the minimum inhibitoryconcentration (MIC) for compound A.

Qb=Concentration of compound B in parts per million, acting alone, whichproduced an end point. This represents the minimum inhibitoryconcentration (MIC) for compound B.

QA=Concentration of compound A in parts per million, in the mixture,which produced an end point.

QB=Concentration of compound B in parts per million, in the mixture,which produced an end point.

When the synergism index is greater than one, antagonism is indicatedand when the index is equal to one, additivity is indicated. When theindex is less than one, synergism exits.

Table 1 below shows the synergism indices as calculated using the Kullmethod for TCMTB (compound A), dodecyl morpholine (DDM, compound B) andTCMTB/DDM combinations against a mixture of fungal species isolated frommolded chrome-tanned leather. The most effective combination tested wasfound to be 2 ppm Busan® 30L and 6 ppm of BL 2180 product, a 60% DDMformulation.

TABLE 1 A: Busan ® B: Dodecyl 30L Morpholine Synergism (30% TCMTE) (60%DDM) Index  4*  0* 1 3 2 0.75 2 6 0.5 2 10 0.5 1.75 60 0.4375 0 80 1*ppm of formulated preparation

EXAMPLE 2

Bactericidal Activity of TCMTB and an N-Alkyl Heterocyclic Compound

The bactericidal effects of the combined use of TCMTB and N-alkylheterocyclic compounds against bacteria were measured using an 18 HourStokes Broth Test. Combinations of TCMTB and N-dodecyl morpho line (DDM)and of TCMTB and N-dodecyl imidazole (DDI) were tested against thebacteria Sphaerotilus natans, ATCC 15291. In this test, compound A wasBusan® 1030 product, a 30% TCMTB formulation, and compound B was eitherDDM (99%) or DDI (99%). The Busan® 30L product is available from BuckmanLaboratories, Memphis, Tenn.

The Stokes broth substrate used to evaluate effectiveness ofmicrobicidal compositions against Sphaerotilus natans was prepared byadding the following to one liter of water:

Compound g/L Peptone 1.0 Glucose 1.0 MgSO₄.7H₂O 0.2 CaCl₂ 0.05FeCl₃.6H₂O 0.01

The broth was adjusted to pH 7.0 with sodium hydroxide. Forty gramportions were then added to 180 mL Pyrex milk dilution bottles fittedwith Escher rubber stoppers and then sterilized. Each of the followingsubstances was then added to each bottle in the order listed.

-   -   1. Sterile distilled water as required in each individual case        to bring the total weight of the contents of each bottle to 50 g        after all subsequent additions specified below (including        inoculation with the aqueous suspension of test organism) have        been made.    -   2. Solution of toxicant or control agent to be evaluated in such        individual volumes as to give the concentration desired in parts        per million by weight.    -   3. One mL of a 24-hour old S. natans culture grown in Stokes        broth.

After the inoculant suspension of the test organism was added to thebottles they were incubated at 28° C. for 18 hours. After thisincubation, a 1 mL of sample was withdrawn from each bottle, diluted,plated on Stokes agar, and incubated for 72-96 hr. at 28-30° C. Stokesagar was prepared like the broth with 12.5 g agar added per liter. Thenumber of colonies on each plate was determined and converted to thecount per milliliter of substrate.

From these data, the percentage kills were calculated. The differencebetween the count for the control substrate (with no toxicant) and thecount obtained from the substrate containing toxicant was divided by thecount for the control substrate to give the fraction killed, which wasthen converted to “percentage killed” by multiplying by 100. The resultsare shown in Tables 2 and 3. A percentage kill of 90 percent or higherrepresents an extremely useful bactericidal composition. Synergismindices were calculated for some combinations using the Kull method asdescribed in Example 1. Others may be calculated from the datapresented.

TABLE 2 A: Busan ® 1030 (30% B: Dodecyl Synergism TCMTE) MorpholineCFU/mL % Kill Index  0* 0 3.80 × 10⁶ — 1 0 1.13 × 10⁶ 70.3 2 0 1.09 ×10⁶ 71.3 5 0 7.82 × 10⁵ 79.4 10 0 <10³ <99.9 20 0 <10³ <99.9 0 1 1.58 ×10⁶ 58.4 0 2 1.24 × 10⁶ 67.4 0 5 8.51 × 10⁵ 77.6 0 10 <10³ <99.9 0 20<10³ <99.9 1 1 1.02 × 10⁶ 73.2 1 2 6.33 × 10⁵ 83.3 1 5 2.17 × 10⁴ 99.40.60 1 10 <10³ <99.9 2 1 1.14 × 10⁵ 97.0 0.30 2 2 1.85 × 10⁵ 95.1 0.40 25 6.41 × 10⁵ 83.1 2 10 <10³ <99.9 5 1 1.23 × 10⁵ 96.8 0.60 5 2 8.74 ×10⁴ 97.7 0.70 5 5 <10³ <99.9 1.00 5 10 <10³ <99.9 10 1 <10³ <99.9 10 2<10³ <99.9 10 5 <10³ <99.9 10 10 <10³ <99.9 *ppm of formulatedpreparation

TABLE 3 A: Busan ® 1030 B: Dodecyl Synergism (30% TCMTE) ImidazoleCFU/mL % Kill Index  0* 0 5.05 × 10⁶ — 0.2 0 2.63 × 10⁶ 47.9 0.5 0 1.97× 10⁶ 61.0 1 0 1.17 × 10⁶ 76.8 2 0 1.06 × 10⁶ 79.0 5 0 8.20 × 10⁵ 83.810 0 <10³ <99.9 20 0 <10³ <99.9 0 1 1.26 × 10⁶ 75.1 0 2 6.80 × 10⁵ 86.50 5 <10³ <99.9 0 10 <10³ <99.9 0 20 <10³ <99.9 0 50 <10³ <99.9 0 100<10³ <99.9 0.2 2 1.04 × 10⁶ 79.4 0.2 5 <10³ <99.9 1.02 0.2 10 <10³ <99.90.2 20 <10³ <99.9 0.5 1 8.40 × 10⁵ 83.4 0.5 2 5.00 × 10⁵ 90.1 0.45 0.5 5<10³ <99.9 0.5 10 <10³ <99.9 0.5 20 <10³ <99.9 1 1 3.80 × 10⁵ 92.5 0.301 2 7.70 × 10⁵ 84.8 1 5 1.25 × 10⁶ 75.2 1 10 <10³ <99.9 1 20 <10³ <99.92 1 3.50 × 10⁶ 93.1 0.40 2 2 6.50 × 10⁵ 87.1 2 5 <10³ <99.9 2 10 <10³<99.9 *ppm of formulated preparation

EXAMPLE 3

Bactericidal Activity of TCMTB and an N-Alkyl Heterocyclic Compound

The bactericidal effects of the combined use of TCMTB and N-alkylheterocyclic compounds against bacteria were measured using an 28 DayAPI Test. Combinations of TCMTB (compound A) and N-dodecyl morpholine(DDM, compound B) and of TCMTB (compound A) and N-dodecyl imidazole(DDI, compound B) were tested against the bateria Desulfovibriodesulfuricans, ATCC 7757. Busan® 1030 product, a 30% TCMTB formulation,DDM (99%), and DDI (99%) were used in this testing. The Busan® 30Lproduct is available from Buckman Laboratories, Memphis Tenn.

The medium used was a modified API Broth having the followingcomposition:

Compound g/L Yeast extract 5.0 Ascorbic acid 0.1 Sodium lactate 5.2MgSO₄.7H₂O 0.2 K₂HPO₄ 0.6 Ferrous ammonium sulfate 0.1 NaCl 10.0

The compounds were dissolved in 1.0 L of deionized water to form themedium. The pH of the medium was adjusted to a final pH of 6.9 with 1 Nsodium hydroxide prior to sterilization.

The medium was sterilized at 121° C. for 20 minutes and then allowed tocool to room temperature. The medium is then inoculated by adding 10 mlper liter of a 24-hr. broth culture of Desulfovibrio desulfuricans.

From stock solutions of the chemicals to be tested, sufficient amountsof the compounds were added to 1 oz. glass bottles so that when a bottlewas completely filled, the desired concentration of the chemical waspresent. The bottles were then completely filled with the inoculated APIbroth (10 mL innoculum per liter medium) in a manner such as to excludeall air and provide an anaerobic state. The bottles were capped tightlyand inverted gently to mix the contents and, at the same time, to makesure no air bubbles were present.

Incubation was carried out at 37° C. for 28 days. Growth was indicatedby blackening of the culture medium, whereas those bottles havingsufficient chemical to inhibit growth would remain clear. Observationsof growth were made at the end of 7 days, 14 days, 21 days and 28 days.Results were reported using a scale of 0 to 4, where 0=no growth and4=heavy growth.

The results are shown in Tables 4 and 5. The minimum inhibitoryconcentration (MIC) was defined as the lowest concentration tested whichgave no growth in the bottle (clear liquid). Synergism indices werecalculated for some combinations using the Kull method as described inExample 1. Others may be calculated from the data presented.

TABLE 4 A: Busan ® 1030 (30% B: Dodecyl Day Day Synergism TCMTE)Morpholine 7 14 Day 21 Day 28 Index  0* 0 4 4 4 4 10 0 4 4 4 4 20 0 0 23 4 40 0 0 0 0 0 80 0 0 0 0 0 160 0 0 0 0 0 0 10 4 4 4 4 0 20 4 4 4 4 040 2 3 4 4 0 80 0 1 3 4 0 160 0 0 2 3 0 320 0 0 0 0 5 10 4 4 4 4 5 20 44 4 4 5 40 4 4 4 4 5 80 0 2 4 4 5 160 0 0 0 0 0.63 10 10 4 4 4 4 10 20 23 4 4 10 40 1 2 3 4 10 80 0 0 0 0 0.50 10 160 0 0 0 0 20 10 4 4 4 4 2020 0 0 0 0 0.56 20 40 0 0 0 0 20 80 0 0 0 0 20 160 0 0 0 0 40 10 0 0 0 040 20 0 0 0 0 40 40 0 0 0 0 40 80 0 0 0 0 40 160 0 0 0 0 *ppm offormulated preparation

TABLE 5 A: Busan ® 1030 (30% B: Dodecyl Day Day Synergism TCMTE)Morpholine 7 14 Day 21 Day 28 Index  0* 0 4 4 4 4 10 0 4 4 4 4 20 0 0 23 4 40 0 0 0 0 0 80 0 0 0 0 0 160 0 0 0 0 0 0 1 4 4 4 4 0 2 4 4 4 4 0 44 4 4 4 0 8 2 4 4 4 0 16 0 0 0 0 0 32 0 0 0 0 5 2 4 4 4 4 5 4 4 4 4 4 58 0 0 0 0 0.63 5 16 0 0 0 0 10 1 4 4 4 4 10 2 4 4 4 4 10 4 0 2 3 4 10 80 0 0 0 0.75 10 16 0 0 0 0 10 32 0 0 0 0 20 1 4 4 4 4 20 2 0 2 3 4 20 40 0 0 0 0.75 20 8 0 0 0 0 20 16 0 0 0 0 40 1 0 0 2 4 40 2 0 0 0 0 40 4 00 0 0 40 8 0 0 0 0 40 16 0 0 0 0 *ppm of formulated preparation

1. A microbicidal composition comprising: (a) 2-(Thiocyanomethylthio)benzothiazole (TCMTB) and (b) an N-alkyl heterocyclic compound of theformula:

wherein n varies from 5 to 17, the heterocyclic ring defined by

is a saturated or unsaturated, unsubstituted or substituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O and S and wherein (a) and (b) arepresent in a combined amount effective to control the growth of at leastone microorganism and the amount of N-alkyl heterocyclic compound (b)present potentiates the microbicidal activity of the TCMTB (a).
 2. Amicrobicidal composition according to claim 1, wherein the heterocyclicring is unsaturated.
 3. A microbicidal composition according to claim 2,wherein the heterocyclic ring is substituted with at least onesubstituent selected from the group consisting of a substituted orunsubstituted alkyl group, a substituted or unsubstituted alkenyl group,an amino group, an oxo group, and a halogen.
 4. A microbicidalcomposition according to claim 3, wherein the heterocyclic ring definedby

is a five membered ring.
 5. A microbicidal composition according toclaim 4, wherein n is
 7. 6. A microbicidal composition according toclaim 5, wherein the heterocyclic ring defined by

contains an S heteroatom and an oxo group.
 7. A method for controllingthe growth of microorganisms on a substrate comprising the step ofcontacting a substrate susceptible to the growth of microorganisms with:(a) 2-(Thiocyanomethylthio) benzothiazole (TCMTB) and (b) an N-alkylheterocyclic compound of the formula:

wherein n varies from 5 to 17, the heterocyclic ring defined by

is a saturated or unsaturated, unsubstituted or substituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O and S and wherein (a) and (b) arepresent in a combined amount effective to control the growth of at leastone microorganism and the amount of N-alkyl heterocyclic compound (b)present potentiates the microbicidal activity of the TCMTB (a).
 8. Amethod according to claim 7, wherein the heterocyclic ring isunsaturated.
 9. A method according to claim 8, wherein the heterocyclicring is substituted with at least one substituent selected from thegroup consisting of a substituted or unsubstituted alkyl group, asubstituted or unsubstituted alkenyl group, an amino group, an oxogroup, and a halogen.
 10. A method according to claim 9, wherein theheterocyclic ring defined by

is a five membered ring.
 11. A method according to claim 10, wherein nis
 7. 12. A method according to claim 11, wherein the heterocyclic ringdefined by

contains an S heteroatom and an oxo group.
 13. A method for controllingthe growth of microorganisms on a hide during a leather tanning processcomprising the step of contacting leather with: (a)2-(Thiocyanomethylthio) benzothiazole (TCMTB) and (b) an N-alkylheterocyclic compound of the formula:

wherein n varies from 5 to 17, the heterocyclic ring defined by

is a saturated or unsaturated, unsubstituted or substituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O and S and wherein (a) and (b) arepresent in a combined amount effective to control the growth of at leastone microorganism and the amount of N-alkyl heterocyclic compound (b)present potentiates the microbicidal activity of the TCMTB (a).
 14. Amethod according to claim 13, wherein the heterocyclic ring isunsaturated.
 15. A method according to claim 14, wherein theheterocyclic ring is substituted with at least one substituent selectedfrom the group consisting of a substituted or unsubstituted alkyl group,a substituted or unsubstituted alkenyl group, an amino group, an oxogroup, and a halogen.
 16. A method according to claim 15, wherein theheterocyclic ring defined by

is a five membered ring.
 17. A method according to claim 16, wherein nis
 7. 18. A method according to claim 17, wherein the heterocyclic ringdefined by

contains an S heteroatom and an oxo group.
 19. A liquor for use in aleather-tanning process comprising: (a) 2-(Thiocyanomethylthio)benzothiazole (TCMTB) and (b) an N-alkyl heterocyclic compound of theformula:

wherein n varies from 5 to 17, the heterocyclic ring defined by

is a saturated or unsaturated, unsubstituted or substituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O and S, wherein (a) and (b) are presentin a combined amount effective to control the growth of at least onemicroorganism on the leather and the amount of N-alkyl heterocycliccompound (b) present potentiates the microbicidal activity of the TCMTB(a), and wherein the liquor is selected from the group consisting of apickling liquor, a chrome-tanning liquor, a vegetable tanning-liquor, apost-tan washing liquor, a retanning liquor, a dye liquor, and a fatliquor.
 20. A liquor according to claim 19, wherein the heterocyclicring is unsaturated.
 21. A liquor according to claim 20, wherein theheterocyclic ring is substituted with at least one substituent selectedfrom the group consisting of a substituted or unsubstituted alkyl group,a substituted or unsubstituted alkenyl group, an amino group, an oxogroup, and a halogen.
 22. A liquor according to claim 21, wherein theheterocyclic ring defined by

is a five membered ring.
 23. A liquor according to claim 22, wherein nis
 7. 24. A liquor according to claim 23, wherein the heterocyclic ringdefined by

contains an S heteroatom and an oxo group.
 25. A microbicidalcomposition, comprising: (a) 2 -(Thiocyanomethylthio)benzothiazole(TCMTB), and (b) an N-alkyl heterocyclic compound of the formula:

 wherein n varies from 9 to 15, the heterocyclic ring defined by

 is a saturated or unsaturated, substituted or unsubstituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O, and S, and wherein the TCMTB (a) andthe N-alkyl heterocyclic compound (b) are present in a combined amounteffective to control the growth of at least one microorganism and theamount of the N-alkyl heterocyclic compound (b) present potentiates orsynergistically enhances the microbicidal activity of the TCMTB (a). 26.The microbicidal composition according to claim 25, wherein theheterocyclic ring is unsaturated.
 27. The microbicidal compositionaccording to claim 26, wherein the heterocyclic ring is substituted withat least one substituent selected from the group consisting of asubstituted or unsubstituted alkyl group, a substituted or unsubstitutedalkenyl group, an amino group, an oxo group, and a halogen.
 28. Themicrobicidal composition according to claim 27, wherein the heterocyclicring defined by

is a five membered ring.
 29. The microbicidal composition according toclaim 28, wherein the heterocyclic ring defined by

contains an S heteroatom and an oxo group.
 30. A method for controllingthe growth of microorganisms on a substrate, comprising the step ofcontacting a substrate susceptible to the growth of microorganisms with:(a) 2 -(Thiocyanomethylthio)benzothiazole (TCMTB), and (b) an N-alkylheterocyclic compound of the formula:

 wherein n varies from 9 to 15, the heterocyclic ring defined by:

 is a saturated or unsaturated, substituted or unsubstituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O, and S, and wherein the TCMTB (a) andthe N-alkyl heterocyclic compound (b) are present in a combined amounteffective to control the growth of at least one microorganism and theamount of the N-alkyl heterocyclic compound (b) present potentiates orsynergistically enhances the microbicidal activity of the TCMTB (a). 31.The method according to claim 30, wherein the heterocyclic ring isunsaturated.
 32. The method according of claim 31, wherein theheterocyclic ring is substituted with at least one substituent selectedfrom the group consisting of a substituted or unsubstituted alkyl group,a substituted or unsubstituted alkenyl group, an amino group, an oxogroup, and a halogen.
 33. The method according to claim 32, wherein theheterocyclic ring defined by

is a five membered ring.
 34. The method according to claim 33, whereinthe heterocyclic ring defined by

contains an S heteroatom and an oxo group.
 35. A method for controllingthe growth of microorganisms on a hide during a leather tanning process,comprising the step of contacting the leather with: (a) 2-(Thiocyanomethylthio)benzothiazole (TCMTB), and (b) an N-alkylheterocyclic compound of the formula:

 wherein n varies from 9 to 15, the heterocyclic ring defined by

 is a saturated or unsaturated, substituted or unsubstituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O, and S, and wherein the TCMTB (a) andthe N-alkyl heterocyclic compound (b) are present in a combined amounteffective to control the growth of at least one microorganism and theamount of the N-alkyl heterocyclic compound (b) present potentiates orsynergistically enhances the microbicidal activity of the TCMTB (a). 36.The method according to claim 35, wherein the heterocyclic ring isunsaturated.
 37. The method according of claim 36, wherein theheterocyclic ring is substituted with at least one substituent selectedfrom the group consisting of a substituted or unsubstituted alkyl group,a substituted or unsubstituted alkenyl group, an amino group, an oxogroup, and a halogen.
 38. The method according to claim 37, wherein theheterocyclic ring defined by

is a five membered ring.
 39. The method according to claim 38, whereinthe heterocyclic ring defined by

contains an S heteroatom and an oxo group.
 40. A liquor for use in aleather-tanning process, comprising: (a) 2-(Thiocyanomethylthio)benzothiazole (TCMTB), and (b) an N-alkylheterocyclic compound of the formula:

 wherein n varies from 9 to 15, the heterocyclic ring defined by

 is a saturated or unsaturated, substituted or unsubstituted ring havingfour to eight members, and containing at least one heteroatom selectedfrom the group consisting of N, O, and S, and wherein the TCMTB (a) andthe N-alkyl heterocyclic compound (b) are present, in the liquor in acombined amount effective to control the growth of at least onemicroorganism on the leather, and the amount of the N-alkyl heterocycliccompound (b) present potentiates or synergistically enhances themicrobicidal activity of the TCMTB (a), and wherein the liquor isselected from the group consisting of a pickling liquor, achrome-washing liquor, a vegetable-tanning liquor, a post-tan washingliquor, a retanning liquor, a dye liquor, and a fat liquor.
 41. Theliquor according to claim 40, wherein the heterocyclic ring isunsaturated.
 42. The liquor according to claim 41, wherein theheterocyclic ring is substituted with at least one substituted selectedfrom the group consisting of a substituted or unsubstituted alkyl group,a substituted or unsubstituted alkenyl group, an amino group, an oxogroup, and a halogen.
 43. The liquor according to claim 42, wherein theheterocyclic ring defined by

is a five membered ring.
 44. The liquor according to claim 43, whereinthe heterocyclic ring defined by

contains an S heteroatom and an oxo group.